Search

L-3-Glycerophosphate dehydrogenase (EC 1.1.99.5) was purified from pig brain mitochondria by extraction with deoxycholate, ion-exchange chromatography and (NH4)2SO4 fractionation in cholate, and preparative isoelectric focusing in Triton X-100. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single subunit of mol.wt. 75 000. The enzyme contains non-covalently bound FAD and low concentrations of iron and acid labile sulphide. No substrate reducible e.p.r. signals were detected. The conditions of purification, particularly the isoelectric focusing step, lead to considerable loss of FAD and possibly iron-sulphur centres. It is therefore not possible to decide with certainty whether the enzyme is a flavoprotein or a ferroflavoprotein. The enzyme catalyses the oxidation of L-3-glycerophosphate by a variety of electron acceptors, including ubiquinone analogues. A number if compounds known to inhibit ubiquinone oxidoreduction by other enzymes of the respiratory chain failed to inhibit L-3-glycerophosphate dehydrogenase, except at very high concentrations.

A modified procedure has been developed for the purification of rabbit muscle l-glycerol 3-phosphate dehydrogenase. The product of the preparation satisfies all criteria of homogeneity. Some physical properties of the enzyme have been re-investigated. The results suggest that previous preparations may have been contaminated with significant amounts of heavy-molecular-weight protein.

1. A method is described for extraction and partial purification of mitochondrial l-3-glycerophosphate dehydrogenase from pig brain. 2. By the criteria that have so far been applied, the extraction and purification procedures do not modify the activity of the enzyme towards artificial electron acceptors. 3. The amounts of acid-liberatable flavine and iron in the preparation were measured. 4. A study was made of the effects of various analogues of l-3-glycerophosphate on the activity of the enzyme.